Anyone make their own PCR cloning vector? - (Dec/14/2010 )
Does anyone here make their own PCR cloning vectors? The kits are pretty expensive (you've probably noticed...).
I don't use them anymore... At least not for regular cloning of a specific gene. Adding RE sites to your primers, digesting the PCR product and ligating directly into your target vector saves time and money. What do you need them for?
rsm
Rsm on Wed Dec 15 08:37:25 2010 said:
I don't use them anymore... At least not for regular cloning of a specific gene. Adding RE sites to your primers, digesting the PCR product and ligating directly into your target vector saves time and money. What do you need them for?
rsm
They're for sequencing. I keep getting overlapping sequence for part of one of my exons (possibly two different alleles? - not sure) but the other's worked fine. Nested PCR hasn't helped, so I want to clone the PCR products so I can get distinct reads.
Well, you can use restriction digest of your PCR products also for this purpose... I have no experience with making my own TA cloning vector, sorry. A friend of mine used to do it, but it involved blunting and adding dTTP with TDT and so on. If you take reagents and working time into acount, then buying a kit is not that expensive 
.
rsm
In my former lab, we engineered a pBluescript vector to carry a XcmI restriction site via ligation with a synthetic linker. This linker had some well chosen bp.. So upon digestion with XcmI, one gets a 3' T overhang. Nice and simple. Make a batch and share it with the entire lab.
You can use any vector you desire. When designing a TA vector, the two most commonly used RE sites added are XcmI or AhdI.
pBluescript has a AhdI site in the AmpR gene, so only XcmI could be used.. But a different vector with a different selection marker, would mean a different choice of enzyme to use.
perneseblue on Wed Dec 22 06:29:10 2010 said:
In my former lab, we engineered a pBluescript vector to carry a XcmI restriction site via ligation with a synthetic linker. This linker had some well chosen bp.. So upon digestion with XcmI, one gets a 3' T overhang. Nice and simple. Make a batch and share it with the entire lab.
You can use any vector you desire. When designing a TA vector, the two most commonly used RE sites added are XcmI or AhdI.
pBluescript has a AhdI site in the AmpR gene, so only XcmI could be used.. But a different vector with a different selection marker, would mean a different choice of enzyme to use.
That's great advice. I'm just a poxy PhD student so my time can well be spent saving money like this.
After digesting it, did you have to gel-purify it?